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Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix
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Biomaterials 28 (2007) 3644–3655 www.elsevier.com/locate/biomaterials
Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix Marta B. Evangelistaa,b,c,, Susan X. Hsiongc,d, Rui Fernandese, Paula Sampaioe, Hyun-Joon Kongc, Cristina C. Barriasa, Roberto Salemad, Ma´rio A. Barbosaa,b, David J. Mooneyc, Pedro L. Granjaa,b,d a INEB—Instituto de Engenharia Biome´dica, Laborato´rio de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal Universidade do Porto, Faculdade de Engenharia, Dep. Eng. Metalu´rgica e Materiais, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal c Harvard School of Engineering and Applied Sciences, Laboratory for Cell and Tissue Engineering, 40 Oxford Street Rm. 415, Cambridge, MA 02138, USA d University of Michigan, Department Of Chemical Engineering, Ann Arbor, MI 4810, USA e IBMC—Institute for Molecular and Cell Biology, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal b
Received 4 January 2007; accepted 25 April 2007 Available online 3 May 2007
Abstract There is a need for new therapeutic strategies to treat bone defects caused by trauma, disease or tissue loss. Injectable systems for cell transplantation have the advantage of allowing the use of minimally invasive surgical procedures, and thus for less discomfort to patients. In the present study, it is hypothesized that Arg–Gly–Asp (RGD)-coupled in a binary (low and high molecular weight) injectable alginate composition is able to influence bone cell differentiation in a three-dimensional (3D) structure. Viability, metabolic activity, cytoskeleton organization, ultrastructure and differentiation (alkaline phosphatase (ALP), von Kossa, alizarin red stainings and osteocalcin quantification) of immobilized cells were assessed. Cells within RGD-modified alginate microspheres were able to establish more interactions with the synthetic extracellular matrix as visualized by confocal laser scanning microscope and transmission electron microscopy imaging, and presented a much higher level of differentiation (more intense ALP and mineralization stainings and higher levels of osteocalcin secretion) when compared to cells immobilized within unmodified alginate microspheres. These findings demonstrate that peptides covalently coupled to alginate were efficient in influencing cell behavior within this 3D system, and may provide adequate preparation of osteoblasts for cell transplantation. r 2007 Elsevier Ltd. All rights reserved. Keywords: Osteoblasts; Cell encapsulation; Alginate; RGD peptide; ECM (extracellular matrix); Bone regeneration
1. Introduction Regeneration of bone tissue is a considerable health concern, especially among the elderly. Within the existing problems are the loss of tissue due to trauma, disease or extraction of tumors. Given that life expectancy is increasing, the need for therapeutic solutions for regeneration of tissues is likely to have an increasing impact on Corresponding author. INEB—Instituto de Engenharia Biome´dica, Laborato´rio de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Tel.: +35 12 2607 4982; fax: +35 12 2609 4567. E-mail addresses: [email protected], [email protected] (M.B. Evangelista).
0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2007.04.028
human health and in health care. Hence, new strategies providing better solutions are needed. Osteoblasts or osteoprogenitor cells immobilized within microspheres constitute the approach proposed here for promoting the regeneration of bone tissue. This system is ideally able to provide cells, as well as, cell products including growth factors, cytokines and other extracellular matrix (ECM) molecules to the injured site, which may play essential roles in the regenerative process. The use of microspheres with immobilized cells has already been widely described in the literature, although usually with the aim of providing immunoisolation; for instance, transplantation of islets of Langerhans to [1–3] solve insulin needs of diabetic patients, and hepatic cells [4–6] in
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the case of acute liver disease. However, transplantation of cells for regenerative purposes, using this type of threedimensional (3D) structure has not been approved as a therapy. Osteoblasts immobilized within microspheres potentially constitute an injectable system due to the small size of microspheres, which can be kept in place by filling-in the defect of interest. This system has several advantages such as easy applicability, less discomfort for patients, and thus may provide faster recovery and lower health costs [7]. Osteoblasts are an anchorage-dependent cell population that needs sites of support for survival. The cell recognition sites of proteins present in the ECM, such as fibronectin and vitronectin, interact with integrins present in the cell membrane and allow cell adhesion [8]. This is the key and first event occurring that will influence the following steps in cell behavior [9]. Proliferation and differentiation are directly influenced by the success of this initial event. A variety of materials have been used to immobilize osteoblasts or preosteoblasts for 3D culture or transplantation. Majmudar et al. [10] first immobilized osteoblasts within alginate in 1991 and showed that chick embryo osteoblasts maintained their phenotype when cultured within alginate microspheres for up to 8 months. Payne et al. [11] reported a temporary gelatin-based system for encapsulation of osteoblasts, to protect the cells within a short period of time during crosslinking of the polymeric vehicle in which cells were encapsulated, poly(propylene fumarate). Lee et al. [12] mixed rat calvarial osteoblasts with poly(aldehyde guluronate) (PAG) hydrogels with different degradation rates, and reported that polymers with very rapid degradation (o1 week) did not lead to bone formation in vivo. On the contrary, PAG hydrogels with slower degradation rates promoted bone formation after 9 weeks. Similarly Alsberg et al. [13] demonstrated that the extent of bone formation was highly dependent on the degradation rate of alginate gels. Burdick and Anseth [14] studied a photocrosslinkable system of poly(ethylene glycol) coupled with the Arg–Gly–Asp (RGD) peptide sequence, and reported that a substantial increase in cell attachment, a more organized structure of F-actin, and an increase in cell mineralization with peptide modification. Alginate was the polymer selected for the present study due to its attractive properties [15], including the possibility to promote cell immobilization using very mild conditions and rapid formation of gel microspheres. In addition, while its biodegradability is typically slow, this can be adjusted using specific ratios of different molecular weight polymer chains [16,17]. However, unmodified it does not promote cell adhesion [18]. An alginate hydrogel system presenting cell adhesion peptides has been investigated for bone regeneration [13], but the cell interaction was only characterized by adhesion and proliferation of bone cells (MC3T3-E1 and rat-derived calvarial osteoblasts) adherent to, not in, the alginate gel
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and cell differentiation was analyzed with a limited number of markers. In the present study, detailed in vitro studies were performed on cells immobilized in modified alginate microspheres. The alginate composition was further improved in order to promote polymer biodegradability and immobilized cells’ viability by using a mixture of oxidized high and low molecular weight alginate. It has been previously shown that this combination of different alginate molecular weights and oxidation can lead to gel degradation within a desirable time-frame for bone regeneration [17,19]. The present study addressed the hypothesis that RGDmodified alginate gels, in a binary composition of a high and low molecular weight, will be able to influence a variety of functions of immobilized osteoblasts, including adhesion, proliferation and differentiation. For this study, MC3T3-E1 cells, a preosteoblastic cell line, were immobilized within RGD-coupled alginate microspheres as well as within unmodified alginate microspheres, for up to 29 days, in dynamic culture conditions and cells were analyzed in terms of viability, metabolic activity, cell structure and ultrastructure, proliferation and differentiation. 2. Materials and methods 2.1. Preparation of alginate 2.1.1. Alginate modification Protanal LF 20/40 sodium alginate, with a high content of guluronic acid units, a generous gift from FMC Biopolymers (Oslo, Norway), was used as high molecular weight (high MW) component to prepare alginate gels (Mn ¼ 218,900 g/mol; Mw ¼ 251,000 g/mol). The low molecular weight (low MW) alginate (Mn ¼ 35,800 g/mol; Mw ¼ 51,300 g/mol) was obtained using g-irradiation with a cobalt-60 source at a dose of 5 Mrad as previously described [20]. The first step in alginate preparation, before any chemical modification, consisted of purification of the polymer through dialysis against deionized water for 3 days using a MWCO 3500 membrane (Spectra/Pors), followed by stirring of the solution with 0.5 g of activated charcoal (Fisher) per gram of alginate. Later, the solution was filtered and lyophilized before further modifications. Oxidation of the sodium alginate polymer was carried out as previously published using sodium periodate [21]. Oligopeptides with a sequence of (Glycine)4–Arginine–Glycine–Aspartic Acid–Serine–Proline (abbreviated as G4RGDSP, Commonwealth Biotechnologies, Inc., Richmond, VA, USA) were covalently coupled to the oxidized alginate using the aqueous carbodiimide chemistry as described in detail by Rowley et al. [18]. For this step, 16.7 mg of the peptide were used per gram of alginate polymer, which corresponds to two peptides per alginate polymer chain for high MW alginate. After reaction for 24 h the alginate was purified by dialysis using a MWCO 3500 (Spectra/Pors), and then filtered, lyophilized and stored at 80 1C until further use.
2.1.2. Alginate gel for cell immobilization The composition designated as RGD-modified alginate corresponds to a 2 wt% alginate solution in 0.9 wt% NaCl (Sigma). It was composed of a 50:50 mixture of oxidized and RGD modified alginate (25 wt% high MW, 75 wt% low MW) and unmodified high MW purified alginate. As a control, a 2 wt% high MW purified alginate, termed from now on as unmodified alginate, was used.
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2.2. Cell immobilization and culture within alginate microspheres 2.2.1. Cell culture A passage number below 15 of MC3T3-E1 cells (a generous gift from Professor Renny Franceschi, University of Michigan) was used in this study. The cells were cultured in a-minimum essential medium (a-MEM) with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/ streptomycin (P/S) and kept at 37 1C in a CO2 incubator with 95% CO2:5% air. The cells were cultured in 300 cm2 flasks until 90% confluent, after which they were trypsinized and passaged. The culture medium was changed every 2 days. All the reagents used for cell culture were purchased from Gibco (Invitrogen). 2.2.2. Immobilization of cells within alginate microspheres Cells were recovered with 0.25% trypsin/EDTA solution before reaching confluency, counted using a Z2 Coulters particle analyzer (Beckman Coulter, Fullerton, CA, USA), resuspended in 0.9 wt% NaCl and loaded in a 5 mL syringe. After centrifugation and discarding of the supernatant, cells were carefully homogenized with the 2 wt% RGDmodified alginate in 0.9 wt% NaCl solution using a dual-syringe system. The final density was 20 106 cells/mL of alginate. Subsequently, the alginate-cell suspension was extruded under a coaxial nitrogen-flow using a Var J1 encapsulation unit (Nisco, Switzerland) at a speed of 40 mL/h (Harvard Apparatus 22, Southnatick, MA, USA). The microspheres were allowed to form, under constant stirring, in an isotonic 0.1 M CaCl2 solution, and were kept therein for 10 min. The microspheres were then rinsed 4 times in phosphate buffer saline (PBS) followed by 4 rinses in culture medium. The same procedure was performed using unmodified alginate. Cells immobilized within alginate microspheres (ca. 1 mm +) were cultured in dynamic culture conditions for up to 29 days using 100 mL spinner flasks (Bellco Biotechnologies, Vineland, NJ, USA). The culture medium was changed every other day and supplemented with a final concentration of 50 mg/mL ascorbic acid (Sigma) and 10 mM b-glycerophosphate (Sigma). Microspheres of both types of alginates but without cells were also prepared as a control.
2.3. Cell analysis: viability, metabolic activity, structure and ultrastructure 2.3.1. Cell viability At days 9, 17 and 24 post-immobilization, cells within alginate microspheres were analyzed in terms of viability. Microspheres recovered from the spinner flasks were washed with warm PBS and incubated in 1 mM calcein (Molecular Probes) solution (Ex 495/Em 520 nm) for 20 min. After a washing step with PBS, microspheres were incubated with 1.5 mM propidium iodide (Molecular Probes) solution in PBS (Ex 535/Em 617 nm) for 5 min followed again by a new washing step. Immediately after, the microspheres were visualized in a confocal laser scanning microscope (CLSM) (Leica SP2 AOBS; Leica Microsystems) using LCS software (Leica Microsystems), in a 37 1C environment. Calcein is only actively hydrolyzed in calcein AM by live cells that are characterized by having an intact cytoplasmic membrane. Propidium iodide is incorporated within partially or totally destroyed cells, which only occurs in dead cells. For quantification purposes, at least 10 images of independent microspheres for each condition were tested and quantification was performed using Image J 1.34s software (Wayne Rasband, National Institutes of Health, USA). 2.3.2. Cell metabolic activity Samples of microspheres of the different conditions tested were recovered at days 1, 3, 8, 14, 21 and 29 post-immobilization and were incubated in 400 mL of Dulbecco’s modified culture medium, without phenol red (Gibco), supplemented with 10% FBS and 1% P/S in a 24-well plate. To each well 80 mL of (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2 tetrazolium) (MTS) solution (Promega, Madison, WI, USA) were added. The microspheres were incubated for 3 h at 37 1C, after which 80 mL/well were transferred to a 96-well plate and absorbance was measured at 490 nm with reference to 620 nm in a KC4 Synergy HT (Bio-tek Instruments, Winooski, VT, USA) reader. Qualitative images were acquired using an Olympus IX81 microscope coupled with a DP70 camera and DP controller 2.1.1.183 software (all from Olympus, Japan). 2.3.3. Cell structure F-actin of cells immobilized within alginate microspheres were observed under CLSM at days 6, 16 and 23 post-immobilization. For this purpose, microspheres recovered from spinner flasks were prepared as follows. Firstly, microspheres were washed in warm PBS and fixed for 10 min with 3.7% methanol-free formaldehyde (Polysciences) in PBS. After washing in PBS twice, the cell membrane was permeabilized using 0.1% Triton X-100 (Mallinckrodt OR) in PBS for 5 min and then washed again with PBS. Samples were incubated for 1 h, at room temperature and protected from light, in 10 mg/mL RNAse (Sigma) in 1 wt% bovine serum albumin (BSA)/PBS solution. Microspheres were then incubated for 20 min in a 6.6 mM Alexa Fluors 488 phalloidin (Ex 495/Em 518 nm) (Molecular Probes) solution in 1 wt% BSA/PBS solution to label F-actin present in the cell cytoskeleton. After washing in 1 wt% BSA/PBS solution, cell nuclei were counterstained with 1.5 mM propidium iodide solution in PBS. After the last washing step in 1 wt% BSA/PBS solution the microspheres were mounted in Vectashields and stored at 4 1C protected from light until further observation under CLSM. 2.3.4. Cell ultrastructure Cells immobilized within alginate microspheres were analyzed under transmission electron microscopy (TEM). At days 3, 6, 11, 21 and 28 postimmobilization, microspheres were washed in PBS and fixed in 2 wt% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) for 1 h. After washing in 0.1 M sodium cacodylate buffer for 30 min the microspheres were fixed in 2% (v/v) osmium tetroxide in 0.1 M sodium cacodylate for 1 h followed by another washing step in 0.1 M sodium cacodylate buffer for 30 min. Samples were dehydrated in a gradient series of ethanol solutions as follows: 1 wt% uranile acetate in 50% ethanol for 10 min, followed by 70%, 80%, 90% and 100% (v/v). Inclusion in EPON resin was performed by immersion of microspheres in a gradually increasing series of propylene oxide to EPON as follows: 3:1, 1:1, 1:3 and 0:1 for 30 min each. At the end, inclusion of microspheres in EPON resin was performed in a silicon mold. EPON polymerization took place at 60 1C for 24 h. Sections with 70 nm thickness were prepared using a diamond knife (Diatome, Hatfield, PA, USA) and were recovered to 200 mesh Cu-grids. Staining of sections using 2 wt% uranyl acetate and saturated lead citrate solution, for 7 min each, was performed before observation. Visualization took place at 50 kV in a Zeiss EM 902A microscope (Germany). For each timepoint and condition, four different microspheres were analyzed and two different grids with two sections each were prepared and observed.
2.4. Cell proliferation and differentiation 2.4.1. Cell quantification Microspheres were recovered from the spinner flasks after 1, 3, 8, 14, 21 and 29 days post-immobilization, weighed and dissolved with 55 mM sodium citrate in 0.9% NaCl for 30 min at 37 1C. A cell pellet was obtained by centrifugation at 2000 RPM for 5 min after which cells were resuspended in cell culture medium. The number of cells presented in each sample was counted using a Z2 Coulters particle analyzer. Three replicates per condition were analyzed. 2.4.2. Total DNA quantification The microspheres were allowed to dissolve using 50 mM EDTA in PBS (pH 7.4) for 30 min of incubation at 37 1C. After that, the cell suspension was centrifuged at 10,000 rpm for 5 min at 4 1C (Eppendorf). The cell pellet was resuspended in PBS for washing purposes. After a second centrifugation step,
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2.4.3. Alkaline phosphatase, von Kossa, alizarin red and toluidine blue stainings Microspheres at different timepoints of culture (12, 21 and 28 days) were recovered from dynamic culture conditions and washed with PBS. Afterwards, they were fixed in 4 wt% paraformaldehyde/PBS for 10 min. Dehydration in a gradient series of ethanol was performed and microspheres were embedded in paraffin blocks. Sections of 5 mm thickness were sequentially recovered and different stainings were performed. Before the staining, all sections were dewaxed in xylene and rehydrated in decreasing gradient series of ethanol followed by water. Alkaline phosphatase (ALP) staining was performed using ALP solution for 45 min at room temperature and protection from light. This ALP solution was prepared using a mixture of 80 mL of Naphtol AS-MX phosphate (Sigma kit) and 2 mL of Fast Violet B salt solution (prepared as described in the manufacturer’s instructions). For von Kossa staining sections were incubated in 2.5 wt% silver nitrate (EMD) for 30 min under UV light, followed by incubation in 5 wt% sodium thiosulfate (Aldrich) for 3 min. For alizarin red staining sections were incubated in 2 wt% alizarin red (Sigma) solution (pH 4.3) for 3 min. The toluidine blue staining was performed using 0.1 wt% toluidine blue solution (Fluka) in 30% ethanol for 2 min at room temperature. After staining the slides were washed in deionized and filtered water, with the exception of the alizarin red staining samples, which were washed in 1:1 acetone to xylene solution. With the exclusion of this last staining, all the others were dehydrated in a gradient series of ethanol and xylene. In the end, all slides were mounted with Permounts (Fisher) aqueous mounting medium. At least five independent microspheres were analyzed for each staining. 2.4.4. Osteocalcin (OC) production For analysis of OC synthesized by immobilized cells each cell lysate was used and OC levels were assessed using the Mouse Osteocalcin ELISA kit (Biomedical Technologies, Stoughton, MA, USA) according to manufacturer’s instructions. Absorbance was measured at 450 nm using a KC4 Synergy HT (Bio-tek Instruments, Winooski, VT, USA) reader. The total amount of Osteocalcin was normalized to the amount of DNA present in each sample. Four replicates were analyzed for each condition. 2.4.5. Statistical analysis Cell viability data was analyzed using ANOVA. Data regarding cell metabolic activity, total DNA, total cell number and osteocalcin quantification was analyzed using the Mann–Whitney U test. Results were considered statistically significant when po0.05. Calculations were performed using SPSSs software for Macintosh OSX (version 11.0).
3. Results 3.1. Cell analysis: viability, metabolic activity, structure and ultrastructure 3.1.1. Cell viability Cell viability was evaluated by CLSM using calcein dye (green fluorescence) and propidium iodide (red fluores-
cence) to allow distinction between live and dead cells [24] within the 3D structure of alginate. Fig. 1 illustrates the Z-projection of CLSM images of MC3T3-E1 cells at day 9. Both RGD-modified alginate and unmodified alginates present a high cell viability percentage that was maintained up to 24 days (Fig. 1C). No statistically significant differences between conditions were observed. 3.1.2. Cell metabolic activity The tetrazolium salts used for these studies are bioreduced by metabolically active cells into colored formazan products. Two different types of results are presented (Fig. 2): qualitative data consisting of images from MTS assay of microspheres at days 8 (Fig. 2A and B) and 28 (Fig. 2C and D), and quantitative data (Fig. 2E) showing MTS values normalized by the mass of microspheres analyzed. Qualitative differences between the behavior of cells immobilized within RGD-coupled alginate and unmodified alginate are clear, and metabolic activity is higher for cells immobilized within RGD-modified alginate (Fig. 2A and C),
B
A
C Modified 104 Cell Viability (%)
the cell pellet was resuspended in Passive Lysis buffer (PLB) (Promega) and stored at 20 1C until further quantification. DNA content was determined using the Labarca and Paigen method [22]. Calf thymus DNA was used as a standard (Sigma). Cell lysates previously prepared were thawed in ice and centrifuged. The cell pellets used for DNA quantification were resuspended in cell lysis buffer (25 mM Tris-HCl, 0.4 M NaCl, 0.5% SDS, pH 7.4) described by Caron [23]. The cell lysates were mixed with 0.1 mg/mL bisbenzimide (Hoechst 33258, Sigma) in TNE buffer (10 mM Tris, 0.2 M NaCl, 1 mM EDTA, pH 7.4) and fluorescence was measured using a DyNA Quant 200TM Fluorometer (Amersham Biosciences, San Francisco, CA, USA) that uses Ex 365/Em 460 nm. For each condition and timepoint, four replicates were analyzed.
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Unmodified n.s.
100
n.s.
n.s.
96
92 0
10 20 Days after cell immobilization
30
Fig. 1. MC3T3-E1 cell viability within alginate microspheres. Cells were stained for calcein (green, live cells) and propidium iodide (red, dead cells) at day 9 and imaged at 37 1C. Modified (A) and unmodified (B) alginate microspheres were observed. Original magnification: 200. Percentage of cell viability (C) within modified (represented by a black line) and unmodified (represented by dashed line) alginate microspheres is shown. At least 10 independent microspheres of each condition at each timepoint (day 9, 17 and 24) were analyzed. Values represent mean7SD. n.s. represents no statistical difference between materials.
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Fig. 2. Metabolic activity of MC3T3-E1 cells immobilized within alginate microspheres. At day 28 (modified, C; unmodified, D) the number of cells able to convert MTS salt into formazan is higher than observed at day 8 (modified A; unmodified, B). Quantitative analysis was performed and results were normalized by milligrams of microspheres. Values expressed are means (n ¼ 4)7SD. *pp0.05.
compared to cells immobilized within unmodified alginate (Figs. 2B and D). Moreover, the MTS assay shows that, at almost all timepoints analyzed, there are statistically significant differences between the modified and the unmodified condition, with the exception of day 21 (Fig. 2E). A decrease in the metabolic activity of cells immobilized either with modified or unmodified alginate is also noticeable from day 1 to the later timepoints. 3.1.3. Cell structure F-actin was fluorescently labeled to allow analysis of cytoskeleton organization, as assessed by CLSM. Nuclei in these experiments were counterstained using propidium iodide for better understanding of the cell structure. Fig. 3 presents representative images of cells immobilized within RGD-modified and unmodified alginate microspheres. It is important to underline that, already at day 6, it is possible to find cells with clearly organized F-actin. It can be observed that cells immobilized within RGD-modified alginate were spread, and exhibited elongated actin filaments. In contrast, cells immobilized within unmodified
alginate were round and did not establish points of contact with the alginate extracellular environment, as evidenced by the lack of assemblage of actin filaments. 3.1.4. Cell ultrastructure and ECM synthesis Transmission electron micrographs were recorded at days 3, 6, 11, 21 and 28 of culture. Fig. 4 shows representative data of immobilized cells at days 6 (Fig. 4A and B), 11 (Fig. 4C and D) and 21 (Fig. 4E) corresponding to RGD-modified alginate (Fig. 4A–C and E) and unmodified alginate (Fig. 4D). It is interesting to notice a high number of mitochondria present in cells immobilized within RGD-modified alginate for all the timepoints considered. Rough endoplasmic reticulum (RER) is present over a large area of the cell. Interestingly, large dilatations of RER, named as RER cisternae or dilatations (Fig. 4C and E), are found in all cultures, and are especially present in the RGD-modified condition. The size of these cisternae increases substantially from days 3–21 and to day 28 (some data not shown). Golgi apparatus is present more frequently in
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Fig. 3. Cytoskeleton organization of cells immobilized within modified and unmodified alginate microspheres at days 6 and 23, as observed under CLSM after staining cells with phalloidin (F-actin, green) and propidium iodide (DNA, red). A more organized structure of F-actin is observed when cells were immobilized within modified alginate, as compared to the unmodified condition. Moreover, pictures taken at day 6 show a homogeneous behavior among the cells immobilized within the same condition.
RGD-modified conditions, and is usually associated with RER and in a juxtanuclear position. Secretion of ECM fibers to the ECM, most likely collagen, is found only for the RGD-modified condition (Fig. 4B), starting on day 3 up to day 11 (some data not shown). Well organized collagen bundles can be identified in the crossections. In terms of cell ultrastructure and extracellular environment around immobilized cells, cell filopodia constitute evidence of interaction of cells with the alginate matrix. Only cells immobilized within RGD-modified alginate show these cellular extensions. Furthermore, these interactions seem to be more intense due to their length and extension away from the cell membrane. It is also important to highlight that these cell extensions to the alginate matrix are longer as later culture times are considered. Moreover, in the first days after immobilization, nuclei of immobilized cells, either within RGDmodified or unmodified alginate, are lobular and present irregular shapes, demonstrating that cells within these 3D systems behave differently from what usually is described for 2D cell cultures. Concerning cells immobilized within RGD-modified alginate, by day 11 cell nuclei become circular, in contrast to what is found for the unmodified condition, where regular nuclei were observed only at day 3.
3.2. Cell proliferation and differentiation 3.2.1. Cell proliferation Fig. 5A shows total cell numbers over the culture period. A higher value is found for the RGD-modified alginate condition. There is an increase of the total cell number from day 1 to 3, when a peak in the number of cells is found. It is also evident that the number of cells immobilized within alginate microspheres decreases along the timeframe of the culture. 3.3. Alkaline phosphatase (ALP), von Kossa and alizarin red stainings At day 28, histochemical staining for ALP activity, an early marker for osteoblast differentiation, shows a more intense pink color for cells immobilized within RGD modified alginate microspheres (Fig. 6A) compared to the staining obtained for cells immobilized within unmodified alginate (Fig. 6B). These findings show a high production of this enzyme in the case of cells immobilized within RGD-modified alginate. Von Kossa staining is positive for phosphate or carbonate deposits when a brown color indirectly detected by silver nitrate is found. Analysis of the conditions with cells immobilized within RGD-modified (Fig. 6C) and
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Fig. 4. Ultrastructure of cells immobilized, at different timepoints (day 6—A, B; day 11—C, D; and day 21—E), within modified (A–C and E) and unmodified alginate (D) after staining with uranyl acetate and lead citrate and visualizing using TEM. Cells interaction with the surrounding matrix can be assessed through the presence and increase of cellular extensions towards the alginate as later timepoints are considered (day 11—Fig. C and day 21—Fig. E). At day 6 collagen fibrils occupy a large area in the surroundings of cells immobilized within modified alginate, as shown in Fig. B1. Fig. B2 shows a magnified view of those collagen bundles. Rough endoplasmic reticulum dilatations become larger in size as time progresses (Figs. C and E). A high synthetic activity can be inferred from the large area of the nucleus, the high presence of RER and Golgi apparatus (magnified view in Fig. A2), which all correspond to intracellular structures involved in protein synthesis. Important cell organelles are highlighted. Alg—alginate; Coll—collagen; RERd—RER dilatation; F— filopodia; G—Golgi apparatus; M—mitochondrion; N—nucleus; RER—rough endoplasmic reticulum. Arrow represents the connection between RER cisternae and RER. Original magnification: A1: 7200, A2: 48,000, B1: 7200, B2: 12,000, C: 10,560, D: 7200 and E: 10,560.
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Total cell number/mg
A Modified Unmodified
6000 4000
*
2000 0
1
3 8 14 21 Days after cell immobilization
29
B ng OC/ng DNA/24h
0.160
*
Modified Unmodified
0.120 0.080 * 0.040 * 0.000 10
20 Days after cell immobilization
30
Fig. 5. Total cell number and osteocalcin quantification for cells immobilized within modified and unmodified alginate microspheres. Cell proliferation (A) was normalized per mg of microspheres and osteocalcin secretion (B) was normalized by ng of DNA and over 24 h. Values represent means (n ¼ 3, A; n ¼ 4, B) replicates 7SD. *po0.05.
unmodified (Fig. 6D) alginate suggests that these compounds are present in a much higher extent in the RGDmodified condition. Alizarin red staining, which is specific for calcium detection, shows a more intense staining for the conditions with cells immobilized within RGD-modified alginate (Fig. 6A at day 12, and Fig. 6G at day 28), compared to cells immobilized within unmodified alginate (Fig. 6F at day 21 and Fig. 6H at day 28). Furthermore, it is possible to observe that the cytoplasm and extracellular environment of cells within the RGD-modified alginate are also positively stained, which does not occur within cells immobilized within unmodified alginate. 3.3.1. Osteocalcin production Osteocalcin synthesized by immobilized cells was assessed over the culture at days 14, 21 and 29 and normalized to total DNA, and per day (Fig. 5B). Production of osteocalcin by cells immobilized within RGD-modified alginate is higher than in cells immobilized within unmodified alginate, and there differences were statistically significant. There is a peak of osteocalcin secretion at day 21, both for cells immobilized within RGD-modified and in unmodified alginate. 4. Discussion In the present work, the hypothesis that RGD-modified alginate in a binary composition of high and low MW would be able to influence osteoblast cell function in an
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injectable 3D cell transplantation system was thoroughly investigated. Specific cellular events, such as metabolic activity, alkaline phosphatase activity, osteocalcin production and mineralization, were influenced by the alginate synthetic ECM covalently coupled with RGD peptides. Furthermore, differences between cells immobilized within unmodified and RGD-coupled alginate were also observed in terms of cytoskeleton organization, ECM production and cell ultrastructure, such as RER amount and organization, number of mitochondria and shape of nuclei. Independently of the type of alginate used the percentage of viable immobilized cells maintained high from day 9 to 24 (Fig. 1), suggesting an inward flux of nutrients and sufficient levels of oxygen [25] that could reach the cell population within the alginate microspheres. This is consistent with cell number data, which was kept fairly unchanged during this timeframe. Furthermore, it seems that the outward flux of cell metabolites was also efficient and did not interfere with cell viability. The influx of nutrients and the outflux of biological cell metabolites produced by the immobilized cells is likely due to the small size of the immobilization system used in this study. The diameter of microspheres used in the present study was small (1 mm) compared to most other studies described in the literature, where larger diameters are typically used [15]. In in vivo future applications it is most likely desirable to further decrease the microsphere diameter to maximize nutrient availability. In a similar study, Burdick and Anseth [14] immobilized rat calvarial osteoblasts within RGD-coupled acrylated polyethylene glycol (PEG) and found a ca. 20% decrease in viability over 2 weeks of culture, which seems to indicate that RGD coupled-alginate microspheres prepared in the present study are a suitable cell immobilization system, providing not only cell adhesion sites for anchoragedependent cells, but also providing adequate environmental characteristics for supporting cell viability. Several other systems have been previously used for cell immobilization within alginate or polyelectrolyte complexes based on alginate, although mostly for immunoisolation purposes [26]. It seems noteworthy to observe that most of these systems do not promote cell viability, except when transfected cells were maintained at a high viability over 28 days of culture [27]. For regenerative purposes, which is the main aim of the present study, maintenance of high cell viability over prolonged periods of time is an important prerequisite, which seems to be met by using RGD-coupled alginate microspheres prepared according to the current procedure. The use of the binary composition of the alginate probably contributed to this result, by lowering the viscosity of the polymer solution before gelling and thus the mechanical stress experienced by the cells at the time of incorporation into the gel [28]. The cell density used for immobilization was very high (20 millions of cells/mL of polymer) in this study, compared to other studies (2 million [29] or 5 million [30]). This difference in cell number may contribute to the
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Fig. 6. Histochemical evaluation of immobilized cells within modified (A, C, E and G) and unmodified (B, D, F and H) alginate microspheres. ALP staining (A and B) at day 28, von Kossa (C and D) at day 28 and alizarin red staining at days 12 (E), 21 (F) and 28 (G and H) were performed in 5 mm thickness paraffin sections. Original magnification: 600.
high cell viability with this immobilization system, as it would allow greater cell–cell interactions. Another key factor that may play an important role in the formation of microspheres and function of the immobilized cells is the gelation time. The time required for preparation of microspheres may be as crucial as the material or the cell type used. In the present study the short time used for crosslinking the microspheres in the calcium chloride bath (10 min), in comparison with the values usually used (30 min) [31], can potentially be favorable to the maintenance of a high of cell viability. Cell metabolic activity is kept almost constant during the 29 days of culture (Fig. 2), despite slightly decreasing from
the first day to the following days. The short-term decrease is probably related to the cells requiring adaptation to the new 3D environment represented by microspheres, compared to monolayer cell culture. The same level of metabolic activity is maintained during the remaining timepoints considered, probably because several distinct cellular events have their maximum expression at different timepoints of the culture, thus contributing differently in time to the total measured metabolic activity [32]. For cells immobilized within RGD-modified alginate the measured values are significantly higher than for cells immobilized within unmodified alginate. RGD peptides covalently coupled to the alginate polymer backbone will
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mediate the adhesion of these cells through integrins present in the cytoplasmic membrane [33], which will then trigger different cellular functions such as synthesis of proteins, proliferation, motility and cytoskeleton organization [34,35]. Altogether, this chemical modification is likely to influence the cell machinery and control several interrelated bioprocesses, such as the metabolic activity. Very few related works, concerning the immobilization of bone cells within microspheres, exist in the literature for comparison. Payne et al. [36] immobilized rat bone marrow stromal cells in gelatin microparticles in order to protect them from the aggressive environment of propylene-co-fumarate polymerization, when used as an injectable vehicle. Immobilized cells were protected and viable but no quantitative data on function was provided. Cytoskeleton organization was assessed in this study by labeling F-actin, and differences between unmodified and RGD-coupled alginate were clearly observed (Fig. 3). As previously discussed the covalently coupled peptides are expected to interact with integrins of the cell membrane and initiate a series of intracellular modifications that will lead to the assembly of actin filaments, thus resulting in an elongated cell shape within the RGD-coupled material. Similar results were reported for materials that were modified with the same recognition peptide sequence [14]. On the contrary, cells immobilized within unmodified alginate were shown to have a round shape, indicating no establishment of sites of interaction with the polymer matrix. Cell ultrastructure was also analyzed and data correlates well with the other findings (Fig. 4). It was observed that cells within unmodified alginate microspheres have unusually shaped cell nuclei, and a low number of mitochondria. This is in accordance with the low levels of metabolic activity measured for this condition. In the case of cells immobilized within RGD-modified alginate a high number of mitochondria was found, as well as filopodia extending to the extracellular space, which correlates with results of cytoskeleton organization, where spread and elongated cells were found for this condition. ECM production and secretion are demonstrated by TEM (Fig. 4). Cells immobilized within RGD-modified alginate secreted collagen by as early as day 3. In the case of cells immobilized within unmodified alginate, no collagen was observed. Majmudar [10] reported the synthesis of collagen type I by primary calvarial osteoblasts immobilized within alginate microspheres for 8 weeks. However, in that case, collagen production was only observed after the dissolution of the alginate beads and subsequent culture of cells on tissue culture polystyrene. Collagen production is a key event required for cell differentiation [38] and mineralization [37,39]. The presence of the RGD peptides within the alginate increased collagen production and secretion to the ECM. Moreover, TEM images also showed high levels of RER, as well as RER cisternae, which increased in volume over time, in the
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case of cells immobilized within RGD-coupled alginate. These dilatations are responsible for lipid and protein biosynthesis, as previously reported by several authors [40–42] and correlate with increased collagen production by cells within the modified condition. Histochemical evaluation of immobilized cells (Fig. 6) showed that ALP activity and presence of carbonates, phosphates and calcium compounds, which are direct signals of mineralization of the ECM, are significantly more pronounced when cells were immobilized within RGD-modified alginate. In agreement with the data previously discussed, the more active cells were able to produce and assemble ECM, and most likely collagen, that was then mineralized over time. Data from independent sources concerning matrix mineralization of the condition with cells immobilized in injectable polymer systems is scarce and sometimes contradictory. Burdick and Anseth performed von Kossa staining of cells immobilized within RGD-coupled PEG and reported a high staining intensity when high concentrations of the peptide were utilized [14]. More recently, Trojani et al. [43] used a silated hydroxypropylmethylcelullose hydrogel for 3D culture of primary osteoblasts and cell lines and reported positive ALP and alizarin red stainings. Kneser et al. [44] immobilized primary osteoblasts within a fibrin gel and performed von Kossa staining in tissue sections. They observed mineralization at day 14, as shown by photomicrographs, but no specific [45] detection was performed. Osteocalcin production, a late marker for osteoblasts differentiation, was assessed and a peak at day 21 of the culture was found. These results are similar to previous findings from Alsberg et al. [13] using a similar system for evaluation of differentiation in a 3D system. In agreement with previous results, a remarkable difference was found for osteocalcin production and secretion between RGDmodified and unmodified alginate. It was quantitative and qualitatively demonstrated that the RGD-coupled alginate was able to promote cell differentiation. The maximum expression of osteocalcin was also found by Payne et al. [11] at almost the same timepoint of the culture (day 18). Moreover, Trojani et al. [43] investigated osteocalcin and osteopontin expression by RT-PCR after 3 weeks of immobilization and found a high gene expression for this timepoint.
5. Conclusions Presentation of RGD peptides from the alginate gels described in this report promotes the adhesion and differentiation of MC3T3-E1 preosteoblasts, and injectable gel particles formed from these gels may provide a useful new system for promoting bone regeneration. Further, this system may find broad utility in the transplantation of a variety of other anchorage-dependent cell populations to engineer and regenerate tissues.
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Ackowledgments The authors acknowledge financial support from LusoAmerican Foundation (FLAD, 113/2004), National Institutes of Health (NIH, RO1-DE013033), and Portuguese Foundation for Science and Technology (FCT) (POCI/SAU-BMA/55556/2004) for funding and for awarding M.B.E. a scholarship (SFRH/BD/13354/2003). Authors are also grateful to Dr. Richard Schalek from Center for Nanoscale System at Harvard University (USA) for the preparation of ultrathin sections for TEM experiments and to Prof. Dr. Renny Franceschi (University of Michigan, USA) for kindly providing MC3T3-E1 cells for this study.
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[17] Kong HJ, Alsberg E, Kaigler D, Lee KY, Mooney DJ. Controlling degradation of hydrogels via the size of cross-linked junctions. Adv Mater 2004;16(21):1917–21. [18] Rowley JA, Madlambayan G, Mooney DJ. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 1999;20(1): 45–53. [19] Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney DJ. Degradation of partially oxidized alginate and its potential application for tissue engineering. Biotechnol Prog 2001;17(5):945–50. [20] Kong HJ, Smith MK, Mooney DJ. Designing alginate hydrogels to maintain viability of immobilized cells. Biomaterials 2003;24(22): 4023–9. [21] Bouhadir KH, Hausman DS, Mooney DJ. Synthesis of crosslinked poly(aldehyde guluronate) hydrogels. Polymer 1999;40(12): 3575–84. [22] Labarca C, Paigen K. Simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980;102(2):344–52. [23] Caron JM, Kirschner MW. Autoregulation of tubulin synthesis. Bioessays 1986;5(5):211–6. [24] Poole CA, Brookes NH, Clover GM. Keratocyte networks visualised in the living cornea using vital dyes. J Cell Sci 1993;106:685–91. [25] Li RH, Altreuter DH, Gentile FT. Transport characterization of hydrogel matrices for cell encapsulation. Biotechnol Bioeng 1996;50(4):365–73. [26] Dulieu C, Poncelet D, Neufeld RJ. Encapsulation and immobilization techniques. In: Ku¨htreiber WM, Lanza RP, Chick WL, editors. Cell encapsulation technology and therapeutics. Boston: Birkha¨user; 1999. p. 3–17. [27] Peirone M, Ross CJD, Hortelano G, Brash JL, Chang PL. Encapsulation of various recombinant mammalian cell types in different alginate microcapsules. J Biomed Mater Res 1998;42(4): 587–96. [28] Kong HJ, Kaigler D, Kim K, Mooney DJ. Controlling rigidity and degradation of alginate hydrogels via molecular weight distribution. Biomacromolecules 2004;5(5):1720–7. [29] Boninsegna S, Dal Toso R, Dal Monte R. Alginate microspheres loaded with animal cells and coated by a siliceous layer. J Sol–Gel Sci Technol 2003;26(1–3):1151–7. [30] Rokstad AM, Donati I, Borgogna M, Oberholzer J, Strand BL, Espevik T, et al. Cell-compatible covalently reinforced beads obtained from a chemoenzymatically engineered alginate. Biomaterials 2006;27(27):4726–37. [31] Lu MZ, Lan HL, Wang FF, Chang SJ, Wang YJ. Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng 2000;70(5):479–83. [32] Stein GS, Lian JB. Molecular mechanisms mediating proliferation differentiation interrelationships during progressive development of the osteoblast phenotype. Endocrine Rev 1993;14(4):424–42. [33] Siebers MC, ter Brugge PJ, Walboomers XF, Jansen JA. Integrins as linker proteins between osteoblasts and bone replacing materials. A critical review. Biomaterials 2005;26(2):137–46. [34] Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;110(6):673–87. [35] Giancotti FG, Ruoslahti E. Transduction—integrin signaling. Science 1999;285(5430):1028–32. [36] Payne RG, McGonigle JS, Yaszemski MJ, Yasko AW, Mikos AG. Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 2. Viability of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate). Biomaterials 2002;23(22):4373–80. [37] Franceschi RT, Iyer BS. Relationship between collagen-synthesis and expression of the osteoblast phenotype in MC3T3-E1 cells. J Bone Miner Res 1992;7(2):235–46. [38] Franceschi RT. The developmental control of osteoblast-specific gene expression: role of specific transcription factors and the extracellular matrix environment. Crit Rev Oral Biol Med 1999;10(1): 40–57.
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Biomaterials 28 (2007) 3644–3655 www.elsevier.com/locate/biomaterials
Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix Marta B. Evangelistaa,b,c,, Susan X. Hsiongc,d, Rui Fernandese, Paula Sampaioe, Hyun-Joon Kongc, Cristina C. Barriasa, Roberto Salemad, Ma´rio A. Barbosaa,b, David J. Mooneyc, Pedro L. Granjaa,b,d a INEB—Instituto de Engenharia Biome´dica, Laborato´rio de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal Universidade do Porto, Faculdade de Engenharia, Dep. Eng. Metalu´rgica e Materiais, Rua Dr. Roberto Frias s/n, 4200-465 Porto, Portugal c Harvard School of Engineering and Applied Sciences, Laboratory for Cell and Tissue Engineering, 40 Oxford Street Rm. 415, Cambridge, MA 02138, USA d University of Michigan, Department Of Chemical Engineering, Ann Arbor, MI 4810, USA e IBMC—Institute for Molecular and Cell Biology, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal b
Received 4 January 2007; accepted 25 April 2007 Available online 3 May 2007
Abstract There is a need for new therapeutic strategies to treat bone defects caused by trauma, disease or tissue loss. Injectable systems for cell transplantation have the advantage of allowing the use of minimally invasive surgical procedures, and thus for less discomfort to patients. In the present study, it is hypothesized that Arg–Gly–Asp (RGD)-coupled in a binary (low and high molecular weight) injectable alginate composition is able to influence bone cell differentiation in a three-dimensional (3D) structure. Viability, metabolic activity, cytoskeleton organization, ultrastructure and differentiation (alkaline phosphatase (ALP), von Kossa, alizarin red stainings and osteocalcin quantification) of immobilized cells were assessed. Cells within RGD-modified alginate microspheres were able to establish more interactions with the synthetic extracellular matrix as visualized by confocal laser scanning microscope and transmission electron microscopy imaging, and presented a much higher level of differentiation (more intense ALP and mineralization stainings and higher levels of osteocalcin secretion) when compared to cells immobilized within unmodified alginate microspheres. These findings demonstrate that peptides covalently coupled to alginate were efficient in influencing cell behavior within this 3D system, and may provide adequate preparation of osteoblasts for cell transplantation. r 2007 Elsevier Ltd. All rights reserved. Keywords: Osteoblasts; Cell encapsulation; Alginate; RGD peptide; ECM (extracellular matrix); Bone regeneration
1. Introduction Regeneration of bone tissue is a considerable health concern, especially among the elderly. Within the existing problems are the loss of tissue due to trauma, disease or extraction of tumors. Given that life expectancy is increasing, the need for therapeutic solutions for regeneration of tissues is likely to have an increasing impact on Corresponding author. INEB—Instituto de Engenharia Biome´dica, Laborato´rio de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Tel.: +35 12 2607 4982; fax: +35 12 2609 4567. E-mail addresses: [email protected], [email protected] (M.B. Evangelista).
0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2007.04.028
human health and in health care. Hence, new strategies providing better solutions are needed. Osteoblasts or osteoprogenitor cells immobilized within microspheres constitute the approach proposed here for promoting the regeneration of bone tissue. This system is ideally able to provide cells, as well as, cell products including growth factors, cytokines and other extracellular matrix (ECM) molecules to the injured site, which may play essential roles in the regenerative process. The use of microspheres with immobilized cells has already been widely described in the literature, although usually with the aim of providing immunoisolation; for instance, transplantation of islets of Langerhans to [1–3] solve insulin needs of diabetic patients, and hepatic cells [4–6] in
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the case of acute liver disease. However, transplantation of cells for regenerative purposes, using this type of threedimensional (3D) structure has not been approved as a therapy. Osteoblasts immobilized within microspheres potentially constitute an injectable system due to the small size of microspheres, which can be kept in place by filling-in the defect of interest. This system has several advantages such as easy applicability, less discomfort for patients, and thus may provide faster recovery and lower health costs [7]. Osteoblasts are an anchorage-dependent cell population that needs sites of support for survival. The cell recognition sites of proteins present in the ECM, such as fibronectin and vitronectin, interact with integrins present in the cell membrane and allow cell adhesion [8]. This is the key and first event occurring that will influence the following steps in cell behavior [9]. Proliferation and differentiation are directly influenced by the success of this initial event. A variety of materials have been used to immobilize osteoblasts or preosteoblasts for 3D culture or transplantation. Majmudar et al. [10] first immobilized osteoblasts within alginate in 1991 and showed that chick embryo osteoblasts maintained their phenotype when cultured within alginate microspheres for up to 8 months. Payne et al. [11] reported a temporary gelatin-based system for encapsulation of osteoblasts, to protect the cells within a short period of time during crosslinking of the polymeric vehicle in which cells were encapsulated, poly(propylene fumarate). Lee et al. [12] mixed rat calvarial osteoblasts with poly(aldehyde guluronate) (PAG) hydrogels with different degradation rates, and reported that polymers with very rapid degradation (o1 week) did not lead to bone formation in vivo. On the contrary, PAG hydrogels with slower degradation rates promoted bone formation after 9 weeks. Similarly Alsberg et al. [13] demonstrated that the extent of bone formation was highly dependent on the degradation rate of alginate gels. Burdick and Anseth [14] studied a photocrosslinkable system of poly(ethylene glycol) coupled with the Arg–Gly–Asp (RGD) peptide sequence, and reported that a substantial increase in cell attachment, a more organized structure of F-actin, and an increase in cell mineralization with peptide modification. Alginate was the polymer selected for the present study due to its attractive properties [15], including the possibility to promote cell immobilization using very mild conditions and rapid formation of gel microspheres. In addition, while its biodegradability is typically slow, this can be adjusted using specific ratios of different molecular weight polymer chains [16,17]. However, unmodified it does not promote cell adhesion [18]. An alginate hydrogel system presenting cell adhesion peptides has been investigated for bone regeneration [13], but the cell interaction was only characterized by adhesion and proliferation of bone cells (MC3T3-E1 and rat-derived calvarial osteoblasts) adherent to, not in, the alginate gel
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and cell differentiation was analyzed with a limited number of markers. In the present study, detailed in vitro studies were performed on cells immobilized in modified alginate microspheres. The alginate composition was further improved in order to promote polymer biodegradability and immobilized cells’ viability by using a mixture of oxidized high and low molecular weight alginate. It has been previously shown that this combination of different alginate molecular weights and oxidation can lead to gel degradation within a desirable time-frame for bone regeneration [17,19]. The present study addressed the hypothesis that RGDmodified alginate gels, in a binary composition of a high and low molecular weight, will be able to influence a variety of functions of immobilized osteoblasts, including adhesion, proliferation and differentiation. For this study, MC3T3-E1 cells, a preosteoblastic cell line, were immobilized within RGD-coupled alginate microspheres as well as within unmodified alginate microspheres, for up to 29 days, in dynamic culture conditions and cells were analyzed in terms of viability, metabolic activity, cell structure and ultrastructure, proliferation and differentiation. 2. Materials and methods 2.1. Preparation of alginate 2.1.1. Alginate modification Protanal LF 20/40 sodium alginate, with a high content of guluronic acid units, a generous gift from FMC Biopolymers (Oslo, Norway), was used as high molecular weight (high MW) component to prepare alginate gels (Mn ¼ 218,900 g/mol; Mw ¼ 251,000 g/mol). The low molecular weight (low MW) alginate (Mn ¼ 35,800 g/mol; Mw ¼ 51,300 g/mol) was obtained using g-irradiation with a cobalt-60 source at a dose of 5 Mrad as previously described [20]. The first step in alginate preparation, before any chemical modification, consisted of purification of the polymer through dialysis against deionized water for 3 days using a MWCO 3500 membrane (Spectra/Pors), followed by stirring of the solution with 0.5 g of activated charcoal (Fisher) per gram of alginate. Later, the solution was filtered and lyophilized before further modifications. Oxidation of the sodium alginate polymer was carried out as previously published using sodium periodate [21]. Oligopeptides with a sequence of (Glycine)4–Arginine–Glycine–Aspartic Acid–Serine–Proline (abbreviated as G4RGDSP, Commonwealth Biotechnologies, Inc., Richmond, VA, USA) were covalently coupled to the oxidized alginate using the aqueous carbodiimide chemistry as described in detail by Rowley et al. [18]. For this step, 16.7 mg of the peptide were used per gram of alginate polymer, which corresponds to two peptides per alginate polymer chain for high MW alginate. After reaction for 24 h the alginate was purified by dialysis using a MWCO 3500 (Spectra/Pors), and then filtered, lyophilized and stored at 80 1C until further use.
2.1.2. Alginate gel for cell immobilization The composition designated as RGD-modified alginate corresponds to a 2 wt% alginate solution in 0.9 wt% NaCl (Sigma). It was composed of a 50:50 mixture of oxidized and RGD modified alginate (25 wt% high MW, 75 wt% low MW) and unmodified high MW purified alginate. As a control, a 2 wt% high MW purified alginate, termed from now on as unmodified alginate, was used.
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2.2. Cell immobilization and culture within alginate microspheres 2.2.1. Cell culture A passage number below 15 of MC3T3-E1 cells (a generous gift from Professor Renny Franceschi, University of Michigan) was used in this study. The cells were cultured in a-minimum essential medium (a-MEM) with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/ streptomycin (P/S) and kept at 37 1C in a CO2 incubator with 95% CO2:5% air. The cells were cultured in 300 cm2 flasks until 90% confluent, after which they were trypsinized and passaged. The culture medium was changed every 2 days. All the reagents used for cell culture were purchased from Gibco (Invitrogen). 2.2.2. Immobilization of cells within alginate microspheres Cells were recovered with 0.25% trypsin/EDTA solution before reaching confluency, counted using a Z2 Coulters particle analyzer (Beckman Coulter, Fullerton, CA, USA), resuspended in 0.9 wt% NaCl and loaded in a 5 mL syringe. After centrifugation and discarding of the supernatant, cells were carefully homogenized with the 2 wt% RGDmodified alginate in 0.9 wt% NaCl solution using a dual-syringe system. The final density was 20 106 cells/mL of alginate. Subsequently, the alginate-cell suspension was extruded under a coaxial nitrogen-flow using a Var J1 encapsulation unit (Nisco, Switzerland) at a speed of 40 mL/h (Harvard Apparatus 22, Southnatick, MA, USA). The microspheres were allowed to form, under constant stirring, in an isotonic 0.1 M CaCl2 solution, and were kept therein for 10 min. The microspheres were then rinsed 4 times in phosphate buffer saline (PBS) followed by 4 rinses in culture medium. The same procedure was performed using unmodified alginate. Cells immobilized within alginate microspheres (ca. 1 mm +) were cultured in dynamic culture conditions for up to 29 days using 100 mL spinner flasks (Bellco Biotechnologies, Vineland, NJ, USA). The culture medium was changed every other day and supplemented with a final concentration of 50 mg/mL ascorbic acid (Sigma) and 10 mM b-glycerophosphate (Sigma). Microspheres of both types of alginates but without cells were also prepared as a control.
2.3. Cell analysis: viability, metabolic activity, structure and ultrastructure 2.3.1. Cell viability At days 9, 17 and 24 post-immobilization, cells within alginate microspheres were analyzed in terms of viability. Microspheres recovered from the spinner flasks were washed with warm PBS and incubated in 1 mM calcein (Molecular Probes) solution (Ex 495/Em 520 nm) for 20 min. After a washing step with PBS, microspheres were incubated with 1.5 mM propidium iodide (Molecular Probes) solution in PBS (Ex 535/Em 617 nm) for 5 min followed again by a new washing step. Immediately after, the microspheres were visualized in a confocal laser scanning microscope (CLSM) (Leica SP2 AOBS; Leica Microsystems) using LCS software (Leica Microsystems), in a 37 1C environment. Calcein is only actively hydrolyzed in calcein AM by live cells that are characterized by having an intact cytoplasmic membrane. Propidium iodide is incorporated within partially or totally destroyed cells, which only occurs in dead cells. For quantification purposes, at least 10 images of independent microspheres for each condition were tested and quantification was performed using Image J 1.34s software (Wayne Rasband, National Institutes of Health, USA). 2.3.2. Cell metabolic activity Samples of microspheres of the different conditions tested were recovered at days 1, 3, 8, 14, 21 and 29 post-immobilization and were incubated in 400 mL of Dulbecco’s modified culture medium, without phenol red (Gibco), supplemented with 10% FBS and 1% P/S in a 24-well plate. To each well 80 mL of (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2 tetrazolium) (MTS) solution (Promega, Madison, WI, USA) were added. The microspheres were incubated for 3 h at 37 1C, after which 80 mL/well were transferred to a 96-well plate and absorbance was measured at 490 nm with reference to 620 nm in a KC4 Synergy HT (Bio-tek Instruments, Winooski, VT, USA) reader. Qualitative images were acquired using an Olympus IX81 microscope coupled with a DP70 camera and DP controller 2.1.1.183 software (all from Olympus, Japan). 2.3.3. Cell structure F-actin of cells immobilized within alginate microspheres were observed under CLSM at days 6, 16 and 23 post-immobilization. For this purpose, microspheres recovered from spinner flasks were prepared as follows. Firstly, microspheres were washed in warm PBS and fixed for 10 min with 3.7% methanol-free formaldehyde (Polysciences) in PBS. After washing in PBS twice, the cell membrane was permeabilized using 0.1% Triton X-100 (Mallinckrodt OR) in PBS for 5 min and then washed again with PBS. Samples were incubated for 1 h, at room temperature and protected from light, in 10 mg/mL RNAse (Sigma) in 1 wt% bovine serum albumin (BSA)/PBS solution. Microspheres were then incubated for 20 min in a 6.6 mM Alexa Fluors 488 phalloidin (Ex 495/Em 518 nm) (Molecular Probes) solution in 1 wt% BSA/PBS solution to label F-actin present in the cell cytoskeleton. After washing in 1 wt% BSA/PBS solution, cell nuclei were counterstained with 1.5 mM propidium iodide solution in PBS. After the last washing step in 1 wt% BSA/PBS solution the microspheres were mounted in Vectashields and stored at 4 1C protected from light until further observation under CLSM. 2.3.4. Cell ultrastructure Cells immobilized within alginate microspheres were analyzed under transmission electron microscopy (TEM). At days 3, 6, 11, 21 and 28 postimmobilization, microspheres were washed in PBS and fixed in 2 wt% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) for 1 h. After washing in 0.1 M sodium cacodylate buffer for 30 min the microspheres were fixed in 2% (v/v) osmium tetroxide in 0.1 M sodium cacodylate for 1 h followed by another washing step in 0.1 M sodium cacodylate buffer for 30 min. Samples were dehydrated in a gradient series of ethanol solutions as follows: 1 wt% uranile acetate in 50% ethanol for 10 min, followed by 70%, 80%, 90% and 100% (v/v). Inclusion in EPON resin was performed by immersion of microspheres in a gradually increasing series of propylene oxide to EPON as follows: 3:1, 1:1, 1:3 and 0:1 for 30 min each. At the end, inclusion of microspheres in EPON resin was performed in a silicon mold. EPON polymerization took place at 60 1C for 24 h. Sections with 70 nm thickness were prepared using a diamond knife (Diatome, Hatfield, PA, USA) and were recovered to 200 mesh Cu-grids. Staining of sections using 2 wt% uranyl acetate and saturated lead citrate solution, for 7 min each, was performed before observation. Visualization took place at 50 kV in a Zeiss EM 902A microscope (Germany). For each timepoint and condition, four different microspheres were analyzed and two different grids with two sections each were prepared and observed.
2.4. Cell proliferation and differentiation 2.4.1. Cell quantification Microspheres were recovered from the spinner flasks after 1, 3, 8, 14, 21 and 29 days post-immobilization, weighed and dissolved with 55 mM sodium citrate in 0.9% NaCl for 30 min at 37 1C. A cell pellet was obtained by centrifugation at 2000 RPM for 5 min after which cells were resuspended in cell culture medium. The number of cells presented in each sample was counted using a Z2 Coulters particle analyzer. Three replicates per condition were analyzed. 2.4.2. Total DNA quantification The microspheres were allowed to dissolve using 50 mM EDTA in PBS (pH 7.4) for 30 min of incubation at 37 1C. After that, the cell suspension was centrifuged at 10,000 rpm for 5 min at 4 1C (Eppendorf). The cell pellet was resuspended in PBS for washing purposes. After a second centrifugation step,
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2.4.3. Alkaline phosphatase, von Kossa, alizarin red and toluidine blue stainings Microspheres at different timepoints of culture (12, 21 and 28 days) were recovered from dynamic culture conditions and washed with PBS. Afterwards, they were fixed in 4 wt% paraformaldehyde/PBS for 10 min. Dehydration in a gradient series of ethanol was performed and microspheres were embedded in paraffin blocks. Sections of 5 mm thickness were sequentially recovered and different stainings were performed. Before the staining, all sections were dewaxed in xylene and rehydrated in decreasing gradient series of ethanol followed by water. Alkaline phosphatase (ALP) staining was performed using ALP solution for 45 min at room temperature and protection from light. This ALP solution was prepared using a mixture of 80 mL of Naphtol AS-MX phosphate (Sigma kit) and 2 mL of Fast Violet B salt solution (prepared as described in the manufacturer’s instructions). For von Kossa staining sections were incubated in 2.5 wt% silver nitrate (EMD) for 30 min under UV light, followed by incubation in 5 wt% sodium thiosulfate (Aldrich) for 3 min. For alizarin red staining sections were incubated in 2 wt% alizarin red (Sigma) solution (pH 4.3) for 3 min. The toluidine blue staining was performed using 0.1 wt% toluidine blue solution (Fluka) in 30% ethanol for 2 min at room temperature. After staining the slides were washed in deionized and filtered water, with the exception of the alizarin red staining samples, which were washed in 1:1 acetone to xylene solution. With the exclusion of this last staining, all the others were dehydrated in a gradient series of ethanol and xylene. In the end, all slides were mounted with Permounts (Fisher) aqueous mounting medium. At least five independent microspheres were analyzed for each staining. 2.4.4. Osteocalcin (OC) production For analysis of OC synthesized by immobilized cells each cell lysate was used and OC levels were assessed using the Mouse Osteocalcin ELISA kit (Biomedical Technologies, Stoughton, MA, USA) according to manufacturer’s instructions. Absorbance was measured at 450 nm using a KC4 Synergy HT (Bio-tek Instruments, Winooski, VT, USA) reader. The total amount of Osteocalcin was normalized to the amount of DNA present in each sample. Four replicates were analyzed for each condition. 2.4.5. Statistical analysis Cell viability data was analyzed using ANOVA. Data regarding cell metabolic activity, total DNA, total cell number and osteocalcin quantification was analyzed using the Mann–Whitney U test. Results were considered statistically significant when po0.05. Calculations were performed using SPSSs software for Macintosh OSX (version 11.0).
3. Results 3.1. Cell analysis: viability, metabolic activity, structure and ultrastructure 3.1.1. Cell viability Cell viability was evaluated by CLSM using calcein dye (green fluorescence) and propidium iodide (red fluores-
cence) to allow distinction between live and dead cells [24] within the 3D structure of alginate. Fig. 1 illustrates the Z-projection of CLSM images of MC3T3-E1 cells at day 9. Both RGD-modified alginate and unmodified alginates present a high cell viability percentage that was maintained up to 24 days (Fig. 1C). No statistically significant differences between conditions were observed. 3.1.2. Cell metabolic activity The tetrazolium salts used for these studies are bioreduced by metabolically active cells into colored formazan products. Two different types of results are presented (Fig. 2): qualitative data consisting of images from MTS assay of microspheres at days 8 (Fig. 2A and B) and 28 (Fig. 2C and D), and quantitative data (Fig. 2E) showing MTS values normalized by the mass of microspheres analyzed. Qualitative differences between the behavior of cells immobilized within RGD-coupled alginate and unmodified alginate are clear, and metabolic activity is higher for cells immobilized within RGD-modified alginate (Fig. 2A and C),
B
A
C Modified 104 Cell Viability (%)
the cell pellet was resuspended in Passive Lysis buffer (PLB) (Promega) and stored at 20 1C until further quantification. DNA content was determined using the Labarca and Paigen method [22]. Calf thymus DNA was used as a standard (Sigma). Cell lysates previously prepared were thawed in ice and centrifuged. The cell pellets used for DNA quantification were resuspended in cell lysis buffer (25 mM Tris-HCl, 0.4 M NaCl, 0.5% SDS, pH 7.4) described by Caron [23]. The cell lysates were mixed with 0.1 mg/mL bisbenzimide (Hoechst 33258, Sigma) in TNE buffer (10 mM Tris, 0.2 M NaCl, 1 mM EDTA, pH 7.4) and fluorescence was measured using a DyNA Quant 200TM Fluorometer (Amersham Biosciences, San Francisco, CA, USA) that uses Ex 365/Em 460 nm. For each condition and timepoint, four replicates were analyzed.
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Unmodified n.s.
100
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n.s.
96
92 0
10 20 Days after cell immobilization
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Fig. 1. MC3T3-E1 cell viability within alginate microspheres. Cells were stained for calcein (green, live cells) and propidium iodide (red, dead cells) at day 9 and imaged at 37 1C. Modified (A) and unmodified (B) alginate microspheres were observed. Original magnification: 200. Percentage of cell viability (C) within modified (represented by a black line) and unmodified (represented by dashed line) alginate microspheres is shown. At least 10 independent microspheres of each condition at each timepoint (day 9, 17 and 24) were analyzed. Values represent mean7SD. n.s. represents no statistical difference between materials.
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Fig. 2. Metabolic activity of MC3T3-E1 cells immobilized within alginate microspheres. At day 28 (modified, C; unmodified, D) the number of cells able to convert MTS salt into formazan is higher than observed at day 8 (modified A; unmodified, B). Quantitative analysis was performed and results were normalized by milligrams of microspheres. Values expressed are means (n ¼ 4)7SD. *pp0.05.
compared to cells immobilized within unmodified alginate (Figs. 2B and D). Moreover, the MTS assay shows that, at almost all timepoints analyzed, there are statistically significant differences between the modified and the unmodified condition, with the exception of day 21 (Fig. 2E). A decrease in the metabolic activity of cells immobilized either with modified or unmodified alginate is also noticeable from day 1 to the later timepoints. 3.1.3. Cell structure F-actin was fluorescently labeled to allow analysis of cytoskeleton organization, as assessed by CLSM. Nuclei in these experiments were counterstained using propidium iodide for better understanding of the cell structure. Fig. 3 presents representative images of cells immobilized within RGD-modified and unmodified alginate microspheres. It is important to underline that, already at day 6, it is possible to find cells with clearly organized F-actin. It can be observed that cells immobilized within RGD-modified alginate were spread, and exhibited elongated actin filaments. In contrast, cells immobilized within unmodified
alginate were round and did not establish points of contact with the alginate extracellular environment, as evidenced by the lack of assemblage of actin filaments. 3.1.4. Cell ultrastructure and ECM synthesis Transmission electron micrographs were recorded at days 3, 6, 11, 21 and 28 of culture. Fig. 4 shows representative data of immobilized cells at days 6 (Fig. 4A and B), 11 (Fig. 4C and D) and 21 (Fig. 4E) corresponding to RGD-modified alginate (Fig. 4A–C and E) and unmodified alginate (Fig. 4D). It is interesting to notice a high number of mitochondria present in cells immobilized within RGD-modified alginate for all the timepoints considered. Rough endoplasmic reticulum (RER) is present over a large area of the cell. Interestingly, large dilatations of RER, named as RER cisternae or dilatations (Fig. 4C and E), are found in all cultures, and are especially present in the RGD-modified condition. The size of these cisternae increases substantially from days 3–21 and to day 28 (some data not shown). Golgi apparatus is present more frequently in
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Fig. 3. Cytoskeleton organization of cells immobilized within modified and unmodified alginate microspheres at days 6 and 23, as observed under CLSM after staining cells with phalloidin (F-actin, green) and propidium iodide (DNA, red). A more organized structure of F-actin is observed when cells were immobilized within modified alginate, as compared to the unmodified condition. Moreover, pictures taken at day 6 show a homogeneous behavior among the cells immobilized within the same condition.
RGD-modified conditions, and is usually associated with RER and in a juxtanuclear position. Secretion of ECM fibers to the ECM, most likely collagen, is found only for the RGD-modified condition (Fig. 4B), starting on day 3 up to day 11 (some data not shown). Well organized collagen bundles can be identified in the crossections. In terms of cell ultrastructure and extracellular environment around immobilized cells, cell filopodia constitute evidence of interaction of cells with the alginate matrix. Only cells immobilized within RGD-modified alginate show these cellular extensions. Furthermore, these interactions seem to be more intense due to their length and extension away from the cell membrane. It is also important to highlight that these cell extensions to the alginate matrix are longer as later culture times are considered. Moreover, in the first days after immobilization, nuclei of immobilized cells, either within RGDmodified or unmodified alginate, are lobular and present irregular shapes, demonstrating that cells within these 3D systems behave differently from what usually is described for 2D cell cultures. Concerning cells immobilized within RGD-modified alginate, by day 11 cell nuclei become circular, in contrast to what is found for the unmodified condition, where regular nuclei were observed only at day 3.
3.2. Cell proliferation and differentiation 3.2.1. Cell proliferation Fig. 5A shows total cell numbers over the culture period. A higher value is found for the RGD-modified alginate condition. There is an increase of the total cell number from day 1 to 3, when a peak in the number of cells is found. It is also evident that the number of cells immobilized within alginate microspheres decreases along the timeframe of the culture. 3.3. Alkaline phosphatase (ALP), von Kossa and alizarin red stainings At day 28, histochemical staining for ALP activity, an early marker for osteoblast differentiation, shows a more intense pink color for cells immobilized within RGD modified alginate microspheres (Fig. 6A) compared to the staining obtained for cells immobilized within unmodified alginate (Fig. 6B). These findings show a high production of this enzyme in the case of cells immobilized within RGD-modified alginate. Von Kossa staining is positive for phosphate or carbonate deposits when a brown color indirectly detected by silver nitrate is found. Analysis of the conditions with cells immobilized within RGD-modified (Fig. 6C) and
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Fig. 4. Ultrastructure of cells immobilized, at different timepoints (day 6—A, B; day 11—C, D; and day 21—E), within modified (A–C and E) and unmodified alginate (D) after staining with uranyl acetate and lead citrate and visualizing using TEM. Cells interaction with the surrounding matrix can be assessed through the presence and increase of cellular extensions towards the alginate as later timepoints are considered (day 11—Fig. C and day 21—Fig. E). At day 6 collagen fibrils occupy a large area in the surroundings of cells immobilized within modified alginate, as shown in Fig. B1. Fig. B2 shows a magnified view of those collagen bundles. Rough endoplasmic reticulum dilatations become larger in size as time progresses (Figs. C and E). A high synthetic activity can be inferred from the large area of the nucleus, the high presence of RER and Golgi apparatus (magnified view in Fig. A2), which all correspond to intracellular structures involved in protein synthesis. Important cell organelles are highlighted. Alg—alginate; Coll—collagen; RERd—RER dilatation; F— filopodia; G—Golgi apparatus; M—mitochondrion; N—nucleus; RER—rough endoplasmic reticulum. Arrow represents the connection between RER cisternae and RER. Original magnification: A1: 7200, A2: 48,000, B1: 7200, B2: 12,000, C: 10,560, D: 7200 and E: 10,560.
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Total cell number/mg
A Modified Unmodified
6000 4000
*
2000 0
1
3 8 14 21 Days after cell immobilization
29
B ng OC/ng DNA/24h
0.160
*
Modified Unmodified
0.120 0.080 * 0.040 * 0.000 10
20 Days after cell immobilization
30
Fig. 5. Total cell number and osteocalcin quantification for cells immobilized within modified and unmodified alginate microspheres. Cell proliferation (A) was normalized per mg of microspheres and osteocalcin secretion (B) was normalized by ng of DNA and over 24 h. Values represent means (n ¼ 3, A; n ¼ 4, B) replicates 7SD. *po0.05.
unmodified (Fig. 6D) alginate suggests that these compounds are present in a much higher extent in the RGDmodified condition. Alizarin red staining, which is specific for calcium detection, shows a more intense staining for the conditions with cells immobilized within RGD-modified alginate (Fig. 6A at day 12, and Fig. 6G at day 28), compared to cells immobilized within unmodified alginate (Fig. 6F at day 21 and Fig. 6H at day 28). Furthermore, it is possible to observe that the cytoplasm and extracellular environment of cells within the RGD-modified alginate are also positively stained, which does not occur within cells immobilized within unmodified alginate. 3.3.1. Osteocalcin production Osteocalcin synthesized by immobilized cells was assessed over the culture at days 14, 21 and 29 and normalized to total DNA, and per day (Fig. 5B). Production of osteocalcin by cells immobilized within RGD-modified alginate is higher than in cells immobilized within unmodified alginate, and there differences were statistically significant. There is a peak of osteocalcin secretion at day 21, both for cells immobilized within RGD-modified and in unmodified alginate. 4. Discussion In the present work, the hypothesis that RGD-modified alginate in a binary composition of high and low MW would be able to influence osteoblast cell function in an
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injectable 3D cell transplantation system was thoroughly investigated. Specific cellular events, such as metabolic activity, alkaline phosphatase activity, osteocalcin production and mineralization, were influenced by the alginate synthetic ECM covalently coupled with RGD peptides. Furthermore, differences between cells immobilized within unmodified and RGD-coupled alginate were also observed in terms of cytoskeleton organization, ECM production and cell ultrastructure, such as RER amount and organization, number of mitochondria and shape of nuclei. Independently of the type of alginate used the percentage of viable immobilized cells maintained high from day 9 to 24 (Fig. 1), suggesting an inward flux of nutrients and sufficient levels of oxygen [25] that could reach the cell population within the alginate microspheres. This is consistent with cell number data, which was kept fairly unchanged during this timeframe. Furthermore, it seems that the outward flux of cell metabolites was also efficient and did not interfere with cell viability. The influx of nutrients and the outflux of biological cell metabolites produced by the immobilized cells is likely due to the small size of the immobilization system used in this study. The diameter of microspheres used in the present study was small (1 mm) compared to most other studies described in the literature, where larger diameters are typically used [15]. In in vivo future applications it is most likely desirable to further decrease the microsphere diameter to maximize nutrient availability. In a similar study, Burdick and Anseth [14] immobilized rat calvarial osteoblasts within RGD-coupled acrylated polyethylene glycol (PEG) and found a ca. 20% decrease in viability over 2 weeks of culture, which seems to indicate that RGD coupled-alginate microspheres prepared in the present study are a suitable cell immobilization system, providing not only cell adhesion sites for anchoragedependent cells, but also providing adequate environmental characteristics for supporting cell viability. Several other systems have been previously used for cell immobilization within alginate or polyelectrolyte complexes based on alginate, although mostly for immunoisolation purposes [26]. It seems noteworthy to observe that most of these systems do not promote cell viability, except when transfected cells were maintained at a high viability over 28 days of culture [27]. For regenerative purposes, which is the main aim of the present study, maintenance of high cell viability over prolonged periods of time is an important prerequisite, which seems to be met by using RGD-coupled alginate microspheres prepared according to the current procedure. The use of the binary composition of the alginate probably contributed to this result, by lowering the viscosity of the polymer solution before gelling and thus the mechanical stress experienced by the cells at the time of incorporation into the gel [28]. The cell density used for immobilization was very high (20 millions of cells/mL of polymer) in this study, compared to other studies (2 million [29] or 5 million [30]). This difference in cell number may contribute to the
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Fig. 6. Histochemical evaluation of immobilized cells within modified (A, C, E and G) and unmodified (B, D, F and H) alginate microspheres. ALP staining (A and B) at day 28, von Kossa (C and D) at day 28 and alizarin red staining at days 12 (E), 21 (F) and 28 (G and H) were performed in 5 mm thickness paraffin sections. Original magnification: 600.
high cell viability with this immobilization system, as it would allow greater cell–cell interactions. Another key factor that may play an important role in the formation of microspheres and function of the immobilized cells is the gelation time. The time required for preparation of microspheres may be as crucial as the material or the cell type used. In the present study the short time used for crosslinking the microspheres in the calcium chloride bath (10 min), in comparison with the values usually used (30 min) [31], can potentially be favorable to the maintenance of a high of cell viability. Cell metabolic activity is kept almost constant during the 29 days of culture (Fig. 2), despite slightly decreasing from
the first day to the following days. The short-term decrease is probably related to the cells requiring adaptation to the new 3D environment represented by microspheres, compared to monolayer cell culture. The same level of metabolic activity is maintained during the remaining timepoints considered, probably because several distinct cellular events have their maximum expression at different timepoints of the culture, thus contributing differently in time to the total measured metabolic activity [32]. For cells immobilized within RGD-modified alginate the measured values are significantly higher than for cells immobilized within unmodified alginate. RGD peptides covalently coupled to the alginate polymer backbone will
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mediate the adhesion of these cells through integrins present in the cytoplasmic membrane [33], which will then trigger different cellular functions such as synthesis of proteins, proliferation, motility and cytoskeleton organization [34,35]. Altogether, this chemical modification is likely to influence the cell machinery and control several interrelated bioprocesses, such as the metabolic activity. Very few related works, concerning the immobilization of bone cells within microspheres, exist in the literature for comparison. Payne et al. [36] immobilized rat bone marrow stromal cells in gelatin microparticles in order to protect them from the aggressive environment of propylene-co-fumarate polymerization, when used as an injectable vehicle. Immobilized cells were protected and viable but no quantitative data on function was provided. Cytoskeleton organization was assessed in this study by labeling F-actin, and differences between unmodified and RGD-coupled alginate were clearly observed (Fig. 3). As previously discussed the covalently coupled peptides are expected to interact with integrins of the cell membrane and initiate a series of intracellular modifications that will lead to the assembly of actin filaments, thus resulting in an elongated cell shape within the RGD-coupled material. Similar results were reported for materials that were modified with the same recognition peptide sequence [14]. On the contrary, cells immobilized within unmodified alginate were shown to have a round shape, indicating no establishment of sites of interaction with the polymer matrix. Cell ultrastructure was also analyzed and data correlates well with the other findings (Fig. 4). It was observed that cells within unmodified alginate microspheres have unusually shaped cell nuclei, and a low number of mitochondria. This is in accordance with the low levels of metabolic activity measured for this condition. In the case of cells immobilized within RGD-modified alginate a high number of mitochondria was found, as well as filopodia extending to the extracellular space, which correlates with results of cytoskeleton organization, where spread and elongated cells were found for this condition. ECM production and secretion are demonstrated by TEM (Fig. 4). Cells immobilized within RGD-modified alginate secreted collagen by as early as day 3. In the case of cells immobilized within unmodified alginate, no collagen was observed. Majmudar [10] reported the synthesis of collagen type I by primary calvarial osteoblasts immobilized within alginate microspheres for 8 weeks. However, in that case, collagen production was only observed after the dissolution of the alginate beads and subsequent culture of cells on tissue culture polystyrene. Collagen production is a key event required for cell differentiation [38] and mineralization [37,39]. The presence of the RGD peptides within the alginate increased collagen production and secretion to the ECM. Moreover, TEM images also showed high levels of RER, as well as RER cisternae, which increased in volume over time, in the
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case of cells immobilized within RGD-coupled alginate. These dilatations are responsible for lipid and protein biosynthesis, as previously reported by several authors [40–42] and correlate with increased collagen production by cells within the modified condition. Histochemical evaluation of immobilized cells (Fig. 6) showed that ALP activity and presence of carbonates, phosphates and calcium compounds, which are direct signals of mineralization of the ECM, are significantly more pronounced when cells were immobilized within RGD-modified alginate. In agreement with the data previously discussed, the more active cells were able to produce and assemble ECM, and most likely collagen, that was then mineralized over time. Data from independent sources concerning matrix mineralization of the condition with cells immobilized in injectable polymer systems is scarce and sometimes contradictory. Burdick and Anseth performed von Kossa staining of cells immobilized within RGD-coupled PEG and reported a high staining intensity when high concentrations of the peptide were utilized [14]. More recently, Trojani et al. [43] used a silated hydroxypropylmethylcelullose hydrogel for 3D culture of primary osteoblasts and cell lines and reported positive ALP and alizarin red stainings. Kneser et al. [44] immobilized primary osteoblasts within a fibrin gel and performed von Kossa staining in tissue sections. They observed mineralization at day 14, as shown by photomicrographs, but no specific [45] detection was performed. Osteocalcin production, a late marker for osteoblasts differentiation, was assessed and a peak at day 21 of the culture was found. These results are similar to previous findings from Alsberg et al. [13] using a similar system for evaluation of differentiation in a 3D system. In agreement with previous results, a remarkable difference was found for osteocalcin production and secretion between RGDmodified and unmodified alginate. It was quantitative and qualitatively demonstrated that the RGD-coupled alginate was able to promote cell differentiation. The maximum expression of osteocalcin was also found by Payne et al. [11] at almost the same timepoint of the culture (day 18). Moreover, Trojani et al. [43] investigated osteocalcin and osteopontin expression by RT-PCR after 3 weeks of immobilization and found a high gene expression for this timepoint.
5. Conclusions Presentation of RGD peptides from the alginate gels described in this report promotes the adhesion and differentiation of MC3T3-E1 preosteoblasts, and injectable gel particles formed from these gels may provide a useful new system for promoting bone regeneration. Further, this system may find broad utility in the transplantation of a variety of other anchorage-dependent cell populations to engineer and regenerate tissues.
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Ackowledgments The authors acknowledge financial support from LusoAmerican Foundation (FLAD, 113/2004), National Institutes of Health (NIH, RO1-DE013033), and Portuguese Foundation for Science and Technology (FCT) (POCI/SAU-BMA/55556/2004) for funding and for awarding M.B.E. a scholarship (SFRH/BD/13354/2003). Authors are also grateful to Dr. Richard Schalek from Center for Nanoscale System at Harvard University (USA) for the preparation of ultrathin sections for TEM experiments and to Prof. Dr. Renny Franceschi (University of Michigan, USA) for kindly providing MC3T3-E1 cells for this study.
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